Resistance to tamoxifen (TAM), the most often prescribed endocrine treatment for breast cancer, represents a significant problem in the management of the disease. Identification of the key factors involved in the molecular mechanism of TAM resistance will undoubtedly lead to the development of logical therapeutic targets. We have previously reported that stable transfection of protein kinase C alpha (PKCa) into T47D human breast cancer cells results in a hormone-independent phenotype and TAM-resistant tumor growth. Tumors formed from these cells grow in the presence of TAM and regress in the presence of 17Beta-estradiol (E2). Our finding that a pure antiestrogen can inhibit these TAM-resistant tumors may have important implications for the treatment of TAM-resistant breast cancer. This information may now allow us to predict the efficacy of endocrine therapy. For example, tumors overexpressing PKCa may be stimulated to grow if the patient is given TAM therapy, therefore a more appropriate therapy may be an estrogen-like compound or a pure antiestrogen. However our T47D/PKCalpha tumor model cannot determine whether PKCalpha overexpression occurs in patients prior to TAM exposure, or is a result of long-term TAM treatment. The following Specific Aims provide the framework to substantiate the role of PKCalpha as a predictor or as a marker of TAM treatment failure as well as elucidate the downstream genes that may participate in TAM-resistant breast cancer. 1. To determine whether PKCalpha expression is predictive of TAM-treatment failure. We will perform immunohistochemical analysis to determine PKCalpha expression in paraffin-embedded breast cancer specimens obtained from the National Cancer Institute's Cooperative Breast Cancer Tissue Resource (NCI CBCTR). 2. To determine whether PKCa expression increases in recurrent (TAM-resistant) tumors relative to the primary tumor. We propose to study paired biopsies from patients from the Lynn Sage database at NMH where both primary and recurrent paraffin-embedded tumors are available to identify changes, if any, in the intensity and/or frequency of PKCalpha expression by immunohistochemistry. 3. Identify specific genes that are differentially expressed downstream of PKCa that mediate the hormone-independent phenotype in T47D:A18/PKCalpha cells and tumors. To examine the PKCa-mediated signaling pathway we will utilize our T47D:A18/PKCalpha cell culture and tumor model to investigate differential gene expression as determined by Atlas Gene Array technology.